MinION sequencing; Nanopore direct RNA sequencing of S. cerevisiae Mex67-AA, pab1 deltaRRM4 (with rapamycin), W303, 38°C 15min, Pab1 overexpression, biological replicate 2
RNA from yeast cells was extracted using the hot acid phenol method. RNA extraction was performed with the hot phenol method. Briefly, a frozen cell pellet was resuspended in TES-buffer (1% SDS, 5 mM EDTA, 10 mM Tris pH 7.5) and an equal volume of 0.1 M citrate buffered phenol pH 4.3 (Sigma P4682). The mix was incubated at 65 ˚C for 40 min under 1400 rpm shaking, followed by centrifugation at 16 000 x g for 10 min at 4 ˚C. The aqueous phase was extracted again with acidic phenol at 65 ˚C for 20 min, and twice with chloroform at room temperature. RNA was precipitated with ethanol (final 70%) and 60 mM LiCl over-night. The pellet was washed with EtOH and resuspended in water. Yeast polyadenylated RNA for DRS library construction and reverse-transcription qPCR analysis was extracted from 35 μg total RNA using the ThermoFisher dynabeads oligo dT(25) kit (catalog no. 61002 or 61005) according to the manufacturer's protocol. Nanopore direct-RNA libraries were prepared with Direct RNA Sequencing Kit (Oxford Nanopore Technologies, SQK-RNA002) using 150 ng of Saccharomyces cerevisiae oligo(dT)-enriched mRNA, mixed with RNA from other species (human cells). Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD) and basecalled with Guppy (Oxford Nanopore Technologies)